Serpin derived from human hypothalmus

ABSTRACT

The present invention provides nucleotide and amino acid sequences that identify and encode a novel serpin (CAPE) expressed in human hypothalamus. The present invention also provides for antisense molecules to the nucleotide sequences which encode CAPE, expression vectors for the production of purified CAPE, antibodies capable for binding specifically to CAPE, hybridization probes or oligonucleotides for the detection of CAPE-encoding nucleotide sequences, genetically engineered host cells for the expression of Cape, a pharmaceutical composition containing biologically active CAPE, a diagnostic test based on CAPE-encoding nucleic acid molecules, and treatment methods comprising administration of biologically active CAPE.

This application is a divisional application of U.S. application Ser.No. 08/487,823, filed Jun. 7, 1995, now U.S. Pat. No. 5,700,924.

FIELD OF THE INVENTION

The present invention is in the field of molecular biology; moreparticularly, the present invention describes the nucleic acid and aminoacid sequences of a novel serpin expressed in the hypothalamus.

BACKGROUND OF THE INVENTION

Inhibitory Serpins

Serpins are irreversible serine protease inhibitors which areprincipally located extracellularly. As a group, they are defined on thebasis of their structural and functional characteristics: a highmolecular weight (between 370-420 amino acid residues), and a C-terminalreactive region. Proteins which have been assigned to the serpin familyinclude the following: α-1 protease inhibitor, α-1-antichymotrypsin,antithrombin III, α-2-antiplasmin, heparin cofactor II, complement C1inhibitor, plasminogen activator inhibitors 1 and 2, glia derived nexin,protein C inhibitor, rat hepatocyte inhibitors, crmA (a viral serpinwhich inhibits interleukin 1-β cleavage enzyme), human squamous cellcarcinoma antigen which may modulate the host immune response againsttumor cells, human maspin which seems to function as a tumor suppressor,lepidopteran protease inhibitor, leukocyte elastase inhibitor (the onlyknown intracellular serpin), and products from three orthopoxviruses(these products may be involved in the regulation of the blood clottingcascade and/or of the complement cascade in the mammalian host).

Serpins form tight complexes with their target proteases. The serpinregion which binds to the target protease is a mobile, exposed reactivesite loop (RSL) which contains the P1-P1' bond that is cleaved. When thecharacteristic serpin P1-P1' bond cleaves, the serpin structure changesprofoundly, and stability to heat- or guanidine-induced denaturationincreases markedly. These changes are referred to as thestressed-to-relaxed (S→R) transition, and are associated with tightcomplex formation with specific proteases. For the α1-proteinaseinhibitor, cleavage of the P1-P1' bond results in a separation of about69 Å between the two residues (Loebermann H et al (1984) J Mol Biol177:531-556). The ability of a serpin to function as an inhibitor may bedirectly related to its ability to undergo this S→R transition (Bruch Met al (1988) J Biol Chem 263:16626-30; Carrell R W et al (1992) CurrOpin Struct Biol 2:438-446).

In addition, the RSL sequence from P17 to P8 (hinge region) is highlyconserved, and small amino acid with side chains are found at positionsP9, P10, P11, P12, and P15 in active inhibitors. The presence of smallamino acids in this region allows the peptide loop from P14-P2 to beinserted into the middle of the protease inhibitor A-sheet. Theinsertion of this sequence into the A-sheet appears to be important instabilizing the inhibitor, and consequently tightening theprotease/serpin complex. Sequence divergence in the hinge region mayconvert an inhibitor to a substrate.

Noninhibitory Serpins

A number of proteins with no known inhibitory activity are alsocategorized as serpins on the basis of strong sequence and structuralsimilarities. These proteins can be cleaved by specific proteases, butdo not form the tight complexes that inhibit protease activity. Examplesare bird ovalbumin, angiotensinogen, barley protein Z, corticosteroidbinding globulin, thyroxine binding globulin, sheep uterine milkprotein, pig uteroferrin-associated protein, an endoplasmic reticulumheat-shock protein (which binds strongly to collagen and could act as achaperone), pigment epithelium-derived factor, and an estrogen-regulatedprotein from Xenopus.

The nature of the difference between inhibitory and noninhibitoryserpins is not well understood. For example, ovalbumin is unable toundergo this S→R transition (Mottonen et al (1992) Nature 355: 270-273).However, hormone binding globulins, such as thyroxine or cortisolbinding globulins, apparently do undergo the transition from the nativestressed to relaxed conformation upon protease cleavage but do not forma tight complex with specific proteases (Pemberton et al (1988) Nature336: 257-258). The S→R transition may confer an advantage for hormonebinding molecules, and for small molecule binding proteins in general,in that the transition from a stressed to a relaxed conformation mayprovide a method for modulating hormone delivery. Both hormone bindingglobulins have a greater than 30% homology with the archetype of theserpin family, alpha-1-antitrypsin, and sequence matching infers thatthey all share a common secondary and tertiary structure.

Serpins are defined and described in Carrell R and Travis J (1985)Trends Biochem Sci 10:20-24; Carrell R et al (1987) Cold Spring HarborSymp Quant Biol 52:527-535; Huber R and Carrell R W (1989) Biochemistry28:8951-8966; and Remold-O'Donneel E (1993) FEBS Lett 315:105-108.

The novel serpin which is the subject of this application was identifiedamong the cDNAs of a pooled hypothalamus library.

The Hypothalamus

The hypothalamus, the master gland of the human body, is an area ofneuroendocrine cells on the floor and midline of the human brain. It isintimately associated with the nervous system function. The anteriorhypothalamus mostly interacts with parasympathetic pathways, and theposterior with sympathetic. Functionally, the hypothalamus is dividedinto chiasmatic, tuberal and mammillary regions.

The chiasmatic region which develops prenatally has three prominentcomponents, the supraoptic, paraventricular and accessory neurosecretorynuclei; the sexually dimorphic intermediate nucleus (SDN); and thesuprachiasmatic nucleus (SCN). The large supraoptic (SON) andparaventricular neurons (PVN) of the chiasmatic region are unmyelinatedand produce antidiuretic hormone (ADH) and oxytocin; the enzyme,tyrosine hydroxylase; and the monoamine neurotransmitter, dopamine.Subsequently, ADH and oxytocin are stored in the anterior pituitarygland. PVN neurons also produce somatostatin. The neurosecretorygranules of these large-celled neurons produce small amounts ofdynorphin, enkephalins, galanin, cholecystokinin, and neuropeptide Ywhich appear to function as local paracrine agents.

The small accessory neurosecretory neurons produce ADH, tyrosinehydroxylase, neuropeptide Y, and corticotropin releasing hormone (CRH)and have prominent dopamine synapses. Neurons of the chiasmatic regioncontain gamma amino butyric acid (GABA), glutamate, quisqualate,relaxin, melatonin, angiotensin-1, endothelin, N-methyl-D-aspartate(NMDA), neurophysin, and B-adrenergic receptors (Morris J F and Pow D V(1993) Ann NY Acad Sci 689:16-33; Renaud L P et al (1992) Prog Brain Res92:277-288). Projections of all three types of chiasmatic neuronscommunicate with many regions of the central nervous system includingthe brain stem, limbic system, retina and spinal cord.

The sexually dimorphic nucleus (SDN), also known as the intermediatenucleus, is located between the supraoptic and paraventricular nuclei.The SDN appears to be sensitive to steroidal hormones and develops twiceas many cells and is twice as large in males (0.2 mm³) as in females(0.1 mm³) after the age of four. The number of cells decreases withsenescence (at 50 years of age for men and 70, for women); however,cause and effect of associated hormones have not been established.

The superchiasmatic nucleus (SCN) is considered to be the circadianpacemaker of the mammalian brain coordinating both hormonal andbehavioral rhythms. The SCN is sexually dimorphic, elongated in womenand spherical in men; and the number and volume of SCN cells varies withage and season. Biochemical and immunological studies indicate thatserotonin and melanin in concert with G-protein associated/cyclicadenosine monophosphate-linked receptors regulate circadian rhythms(Erlander M G et al (1993) J Biol Rhythms 8S:25-31).

The retinohypothalamic tract is a monosynaptic pathway that links theretina to the SCN and helps set the intrinsic period, phase, andamplitude of the internal biological clock.

Total blindness which prevents light/dark synchronization results infree-running rhythms, particularly in cortisol, melatonin, sleep, andtemperature regulation. A lesion or tumor in the area of the SCN canalso be correlated with disturbed circadian rhythms.

The hypothalamic neurons of the tuberal and mammillary regions produce avariety of regulatory peptides called releasing hormones or factorswhich modulate much of human endocrine function. These shortoligopeptide releasing factors are secreted and delivered to theanterior pituitary via the fenestrated capillary network of thehypothalamic pituitary portal system. Each region will be discussed inturn.

The tuberal region is composed of a complex of ventromedial (VMN),dorsomedial (DMN), lateral tuberal (NTL), and infundibular nuclei(Braak, H and Braak, E (1992) Prog Brain Res 93:3-14). These nucleifunction in feeding, aggressive and sexual behaviors, and they secretegrowth hormone releasing hormone (GRH), thyroid releasing hormone (TRH)and luteinizing hormone releasing hormone (LHRH). The networked VMN hasprojections to the basal forebrain as well as to all parts of thecerebral cortex where it is assumed to influence higher corticalfunction.

The DMN is poorly differentiated in the human brain and covers theanterior and superior areas of the VMN. Its neurons containcatecholamine, somatostatin, neuropeptide Y and neurotensin, neurokininB (NKB), and LHRH. The NKB neurons may participate negative feedback ofestrogen on LHRH and act as an interneuron on LHRH nuclei.

The NTL is only present in higher primates. The NTL is characterized bycholinergic, CRH, somatostatin, benzodiazepin and NMDA receptors.Neuronal loss in this region may predict disease severity, particularlyin Kallmann's and Down's syndromes and in Alzheimer's and Huntington'sdiseases.

The exact role of the mammillary nucleus is poorly defined. Most of itsneurons project into the cortex and are responsible for the majorhistaminergic innervation. Some evidence indicates the mammillary regionis involved in heat regulation and governs capillary restriction,sweating, shivering and piloerection.

Nonendocrine functions of the hypothalamus include regulation of foodintake and feeding-behavior, temperature regulation, sleep-wake cycle,memory, behavior, and thirst. Although the basal hypothalamus is knownto control stable weight, both the VMN and the anterior hypothalamus areinvolved in regulation of hunger and satiety. Appetite is stimulated byGABA, dopamine, beta-endorphins, enkephalin and neuropeptide Y andinhibited by serotonin, norepinephrine, cholecystokinin, neurotensin,TRH, naloxone, somatostatin, and vasoactive intestinal peptide. A lesionor tumor in the area of the VMN can cause hypothalamic obesity. Otherfactors, particularly the thyroid and adrenal hormones, also affecteating behavior.

The anterior hypothalamus contains neurons that respond to local andenvironmental thermal gradients. Heat production is stimulated byserotonin and blocked by norepinephrine and epinephrine. When infectionsoccur, phagocytic cells produce interleukin-1 (IL-1). IL-1 stimulatesthe anterior hypothalamus to produce prostaglandin E2 which increasesthe body temperature set point and produces fever. Cooling or heatdissipation which involves vasodilation is governed by the posteriorhypothalamus. Hypothalamic disease (with or without malfunction of thethyroid or adrenal glands) may cause hypothermia, hyperthermia orpoikilothermia.

The sleep center is located in the anterior hypothalamus wheredisturbances or lesions can lead to insomnia or agitation. The posteriorhypothalamus is responsible for arousal and maintenance of the wakingstate. Serotonin promotes sleep, while catecholamines aid wakefulness.Destruction of the posterior hypothalamus, for example, by ischemia andencephalitis, trauma or tumor can result in hypersomnolence.

Thirst is controlled by serum osmolality and is detected byosmoregulators in the hypothalamus. Nerve impulses control the pituitaryrelease of vasopressin which acts upon the kidney. In the case ofpathological disturbances, interactions among the nervous system,endocrine hormones, and cytokines (particularly IL-1) modulate theactivity of these glands and the kidney. Impaired thirst is commonlyattributable to hypothalamic lesions.

The effects of ACTH, ADH, and oxytocin memory and behavior are stillbeing investigated. Lesions of the ventromedial hypothalamus producerage while ventromedial, dorsomedial and/or mammillary lesions causeloss of short term memory. Lateral hypothalamic destruction can causeapathetic behavior, but large hypothalamic lesions are associated withdementia.

Diseases Associated with the Hypothalamus

Many diseases are associated with changes in hypothalamic function andstructure. The most common hypothalamic disease is hyperprolactinemia,excess prolactin production, which may lead to galactorrhea and/orhypogonadism. Another disease is dwarfism, likely caused by theoverproduction of somatostatin which prevents growth hormone release.Tumors are the most common cause of the over- or under-production ofhypothalamic hormones. Cushing's disease is caused by tumorsoverproducing ACTH. Finally, the hypothalamus or the molecules itproduces may also be responsible for some of the symptoms inneurodegenerative diseases such as Alzheimer's, Parkinson's, andHuntington's diseases.

Hypothalamic anatomy, physiology, and diseases are reviewed, inter alia,in Guyton AC (1991) Textbook of Medical Physiology, W B Saunders Co,Philadelphia Pa.; Isselbacher K J et al (1994) Harrison's Principles ofInternal Medicine, McGraw Hill, New York City; The Merck Manual ofDiagnosis and Therapy (1992) Merck Research Laboratories, Rahway N.J.;and Swaab D B et al (1993) Anat Embryol 187:317-330.

Some of these diseases may be difficult to diagnose or treat. Moderntechniques for diagnosis of abnormalities in the hypothalamus mainlyrely on observation of clinical symptoms, serological analysis ofhormone levels, or measurement of urinary excretion of a hormone or itsmetabolites. Alternatively, computerized axial tomography (CAT scan) orMagnetic Resonance Imaging (MRI) can be used to observe abnormalhistological changes of the hypothalamic region. Thus, development ofnew techniques becomes necessary for early and accurate diagnosis or fortreatments of diseases associated with the hypothalamus.

SUMMARY OF THE INVENTION

The subject invention provides a unique nucleotide sequence (cape) whichencodes a novel serpin (CAPE). The nucleotide sequence, which wasidentified from Incyte Clone 84476 derived from hypothalamic cells,contains two ATG codons downstream from the last stop codon of theprevious gene. The two ATGs predict the expression of two differentproteins: CAPE1 and CAPE2. However only CAPE1 includes a good signalsequence.

The subject invention includes the antisense DNA of cape; cloning orexpression vectors containing cape; host cells or organisms transformedwith expression vectors containing cape; a method for the production andrecovery of purified CAPE polypeptide from host cells; purified CAPEpolypeptide; antibodies to both polypeptides; and pharmacologicalcompounds using CAPE for the treatment of disease.

Furthermore, the subject invention also comprises diagnostic tests forpathologically compromised brain tissues including but not limited tothe hypothalamus which include the steps of testing a sample or anextract thereof with cape DNA, fragments or oligomers thereof.

DESCRIPTION OF THE FIGS.

FIGS. 1A and 1B show the nucleotide sequence for cape including theentire coding sequence and the predicted amino acid sequences for CAPE1and CAPE2 polypeptides. The start codon for CAPE1 is at nucleotide 79;whereas the start codon for CAPE2 is at nucleotide 121.

FIGS. 2A, 2B, 2C, and 2D display the alignment of CAPE1 and CAPE2,respectively, with plasminogen activator inhibitor 2 (PAI-2). Themajority sequence is a consensus sequence. Alignments shown wereproduced using the multisequence alignment program of DNASTAR software(DNASTAR Inc, Madison Wis.).

FIG. 3 provides structural analysis of the cape sequence for determiningputative alpha (A), beta (B), turn (T), and coil (C) regions; ahydrophilicity plot (H); alpha and beta amphipathic regions (^(*));flexible regions (F); a putative antigenic index (Al); and a surfaceprobability plot (S) using the structural analysis program of DNASTARsoftware (DNASTAR Inc, Madison Wis.).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, CAPE1 and CAPE2 refer to novel serpins, naturallyoccurring, or active fragments thereof, which are encoded by mRNAstranscribed from the cDNA (cape) of SEQ ID NO:1. The amino acid sequenceof CAPE1 is shown in SEQ ID NO:2 starting at residue 27 and terminatingat residue 519, and that of CAPE2 is shown in SEQ ID NO:2 starting atresidue 41 and terminating at residue 519. The abbreviation CAPE will beused to describe CAPE1 and CAPE2 generally.

"Active" refers to those forms of CAPE which retain biologic and/orimmunologic activities of any naturally occurring CAPE.

"Naturally occurring CAPE" refers to CAPE produced by human cells thathave not been genetically engineered and specifically contemplatesvarious forms arising from post-translational modifications of thepolypeptide, including but not limited to acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.

"Derivative" refers to polypeptides derived from naturally occurringCAPE by chemical modifications such as ubiquitination, labeling (e.g.,with radionuclides, various enzymes, chromogenic or fluorogenic means),pegylation (derivatization with polyethylene glycol), or by insertion(or substitution by chemical synthesis) of amino acids such asornithine, which do not normally occur in human proteins.

"Recombinant variant" refers to any polypeptide differing from naturallyoccurring CAPE by amino acid (amino acid) insertions, deletions, andsubstitutions, created using recombinant DNA techniques. Guidance indetermining which amino acid residues may be replaced, added or deletedwithout abolishing activities of interest, such as protein proteolysis,protease inhibition, or small molecule binding properties, may be foundby comparing the sequence of the particular CAPE with that of homologousinhibitory and noninhibitory serpins and minimizing the number of aminoacid sequence changes made in regions of high homology.

Preferably, amino acid "substitutions" are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, such as the replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, or a threonine witha serine, i.e., conservative amino acid replacements. "Insertions" or"deletions" are typically in the range of about 1 to 5 amino acid. Thevariation allowed may be experimentally determined by systematicallymaking insertions, deletions, or substitutions of amino acid in a CAPEmolecule using recombinant DNA techniques and assaying the resultingrecombinant variants for activity.

A "signal sequence" can direct a polypeptide to a specific location in acell or to a specific destination outside of the cell. Such a sequencemay be naturally present on the polypeptide of the present invention orprovided from heterologous protein sources by recombinant DNAtechniques.

A polypeptide "fragment," "portion," or "segment" is a stretch of aminoacid residues of at least about 5 amino acid, often at least about 7amino acid, typically at least about 9 to 13 amino acid, and, in variousembodiments, at least about 17 or more amino acid. To be active, anyCAPE polypeptide must have sufficient length to display biologic and/orimmunologic activity on their own or when conjugated to a carrierprotein such as keyhole limpet hemocyanin (KLH, Sigma).

"Small molecules" are molecules with a molecular weight under 5000, morepreferably under 2000. The small molecules of particular interest may bederived from the hypothalamus, such as oxytocin, vasopressin, dopamine,neuropeptide Y, somatostatin, or enkephalins. These small molecules maydirectly affect the hypothalamus or other target neuronal tissues, suchas the pituitary gland. Alternatively, the small molecules may bederived from other tissues and affect the hypothalamus. These smallmolecules may include, but are not limited to, molecules such asserotonin, epinephrine, norepinephrine, gamma amino butyric acid,glutamate, or other neurotransmitters or hormones. These small moleculesmay be naturally occurring or synthetically made.

"Conditions associated with altered expression of CAPE" refer tophysiological or pathological changes of the hypothalamus or otherneuronal tissues. Pathological changes include inflammation, disease andtumors.

"Hypothalamic tissue" refers to tissue derived mostly from thehypothalamus, but which may include other tissue from organs thatsurround or are adjacent to the hypothalamus.

"Animal" as used herein may be defined to include human, domestic, oragricultural (cats, dogs, cows, sheep, etc.) or test species (mouse,rat, rabbit, etc.).

An "oligonucleotide" or polynucleotide "fragment", "portion," or"segment" is a stretch of nucleotide residues which is long enough touse in polymerase chain reaction (PCR) or various hybridizationprocedures. Oligonucleotide probes will comprise sequence that isidentical or complementary to a portion of cape where there is little orno identity or complementarity with any known or prior art molecule. Theoligonucleotide probes will generally comprise between about 10nucleotides and 50 nucleotides, and preferably between about 15nucleotides and about 30 nucleotides. Nucleic acid probes compriseportions of the cape sequence having fewer nucleotides than about 6 kb,preferably fewer than about 1 kb. After appropriate testing to eliminatefalse positives, both oligonucleotide and nucleic acid probes may beused to determine whether mRNAs encoding CAPE are present in a cell ortissue or to isolate similar natural nucleic acid sequences fromchromosomal DNA as described by Walsh, et al (1992) PCR Methods Appl1:241-50.

Probes may be derived from naturally occurring or recombinant single- ordouble-stranded nucleic acids or be chemically synthesized. They may belabeled by nick translation, Klenow fill-in reaction, PCR or othermethods well known in the art. Probes of the present invention, theirpreparation and/or labelling are elaborated in Sambrook, et al (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor N.Y., orAusubel, et al (1989) Current Protocols in Molecular Biology, John Wiley& Sons, New York City, both incorporated herein by reference.

Alternatively, recombinant variants encoding these same or similarpolypeptides may be synthesized or selected by making use of the"redundancy" in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsmay also be introduced to modify the properties of the polypeptide,including but not limited to small molecule-binding affinities, orpolypeptide degradation or turnover rate.

The present invention includes purified CAPE polypeptide from natural orrecombinant sources, and cells transformed with recombinant nucleic acidmolecules encoding CAPE. Various methods for the isolation of thepolypeptide may be accomplished by procedures well known in the art. Forexample, such polypeptides may be purified by immunoaffinitychromatography by employing the antibodies provided by the presentinvention. Various other methods of protein purification well known inthe art include those described in Deutscher M (1990) Methods inEnzymology, Vol 182, Academic Press, San Diego Calif.; and Scopes R(1982) Protein Purification: Principles and Practice. Springer-Verlag,New York City, both incorporated herein by reference.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a nucleotide sequence (cape) for a novelserpin, identified in hypothalamic cells. The sequence is provided inSEQ ID NO:1. FIGS. 1A-1C provide the cape nucleotide sequence, and thepolypeptide sequence it encodes. Interestingly, the nucleotide sequencecontains two alternative start sites (ATG codons) downstream from thestop codon of the previous gene. These start sites serve to express twonovel serpins which possess substantial overlap (>95%) in thepolypeptide sequence. One encoded protein (CAPE1) is expressed from anATG codon at nucleotide position 81; its sequence is presented in SEQ IDNO:2 starting at residue 27. The second protein (CAPE2) is expressedfrom an ATG codon at nucleotide position 121; its sequence is presentedin SEQ ID NO:2 starting at residue 41.

FIGS. 2A-2D provide an alignment of CAPE1 and CAPE2, respectively, withplasminogen activation inhibitor-2 (PAI-2), an exemplary serpin familymember. CAPE1 contains a good signal sequence consisting of hydrophobicresidues indicating that it may be selectively transported fromhypothalamic cells to another location such as the pituitary gland. Onthe other hand, CAPE2, like other serpins, may be secreted fromhypothalamic cells. Overall, about 110 out of 406 residues of CAPE2match exactly with those of PAI-2 (about 27% homology). For the reactivesite loop (RSL) residues P10, and P12-16 match exactly, whereas P8, P11,and P17 are substituted by amino acids that are larger by either anextra carbon group, i.e. the presence of threonine versus serine at P11,or a hydroxyl group, i.e. the presence of serine versus alanine at P8.

Since CAPE appears to have an RSL that resembles that of inhibitoryserpins, CAPE may inhibit unidentified proteases within or outside ofcells. Alternatively, CAPE may serve to bind specific small molecules tomaintain higher levels of these molecules inside or outside of a celland to modulate their release. In fact, the name CAPE was selectedbecause the novel serpin of the subject invention may function either tomask protease activity or to sequester small molecules.

In view of the fact that the cape nucleotide sequence has beenidentified in hypothalamic cells, the nucleic acid (cape), polypeptide(CAPE), and antibody to CAPE may be useful in investigations of and theintervention in the normal and abnormal function of the numerousendocrine and nonendocrine functions of the hypothalamus. However, eventhough the cape sequence was found to be expressed in hypothalamic cellsit should not be ruled out that cape may be expressed in other cells,particularly other neuronal or secretory cells.

The nucleotide sequence encoding cape has numerous applications intechniques known to those skilled in the art of molecular biology. Thesetechniques include use as hybridization probes, use in the constructionof oligomers for PCR, use for chromosome and gene mapping, use in therecombinant production of CAPE and use in the generation of anti-senseDNA or RNA, their chemical analogs and the like. Uses of nucleotidesencoding the proteins disclosed herein are exemplary of known techniquesand are not intended to limit their use in any technique known to aperson of ordinary skill in the art. Furthermore, the nucleotidesequences disclosed herein may be used in molecular biology techniquesthat have not yet been developed, provided the new techniques rely onproperties of nucleotide sequences that are currently known, eg, thetriplet genetic code, specific base pair interactions, etc.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of CAPE-encodingnucleotide sequences, some bearing minimal homology to the nucleotidesequence of any known and naturally occurring gene may be produced. Theinvention has specifically contemplated each and every possiblevariation of nucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe nucleotide sequence of naturally occurring CAPE, and all suchvariations are to be considered as being specifically disclosed.

Although the nucleotide sequences which encode CAPE and/or its variantsare preferably capable of hybridizing to the nucleotide sequence ofnaturally occurring CAPE under stringent conditions, it may beadvantageous to produce nucleotide sequences encoding CAPE or itsderivatives possessing a substantially different codon usage. Codons canbe selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic expression host inaccordance with the frequency with which particular codons are utilizedby the host. Other reasons for substantially altering the nucleotidesequence encoding CAPE and/or its derivatives without altering theencoded aa sequence include the production of RNA transcripts havingmore desirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

Nucleotide sequences encoding CAPE may be joined to a variety of othernucleotide sequences by means of well established recombinant DNAtechniques (cf Sambrook J et al. supra). Useful nucleotide sequences forjoining to cape include an assortment of cloning vectors, e.g.,plasmids, cosmids, lambda phage derivatives, phagemids, and the like,that are well known in the art. Vectors of interest include expressionvectors, replication vectors, probe generation vectors, sequencingvectors, and the like. In general, vectors of interest may contain anorigin of replication functional in at least one organism, convenientrestriction endonuclease sensitive sites, and selectable markers for thehost cell.

Another aspect of the subject invention is to provide for cape-specificnucleic acid hybridization probes capable of hybridizing with naturallyoccurring nucleotide sequences encoding CAPE. Such probes may also beused for the detection of similar serpin encoding sequences and shouldpreferably contain at least 50% of the nucleotides from the conservedregion or active site. The hybridization probes of the subject inventionmay be derived from the nucleotide sequences of SEQ ID NO:1 or fromgenomic sequences including promoters, enhancer elements and/or possibleintrons of respective naturally occurring CAPE molecules. Hybridizationprobes may be labeled by a variety of reporter groups, includingradionuclides such as ³² P or ³⁵ S, or enzymatic labels such as alkalinephosphatase coupled to the probe via avidin/biotin coupling systems, andthe like.

Other means of producing specific hybridization probes for cape DNAsinclude the cloning of nucleic acid sequences encoding CAPE or CAPEderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art and are commercially available and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerase as T7 or SP6 RNA polymerase and theappropriate radioactively labeled nucleotides.

It is now possible to produce a DNA sequence, or portions thereof,encoding CAPE and their derivatives entirely by synthetic chemistry,after which the gene can be inserted into any of the many available DNAvectors using reagents, vectors and cells that are known in the art atthe time of the filing of this application. Moreover, syntheticchemistry may be used to introduce mutations into the cape sequences orany portion thereof.

PCR as described in U.S. Pat. Nos. 4,683,195; 4,800,195; and 4,965,188provides additional uses for oligonucleotides based upon the nucleotidesequence which encodes CAPE. Such probes used in PCR may be ofrecombinant origin, may be chemically synthesized, or a mixture of bothand comprise a discrete nucleotide sequence for diagnostic use or adegenerate pool of possible sequences for identification of closelyrelated genomic sequences.

Full length genes may be cloned from known sequence using a new methodwhich employs XL-PCR (Perkin-Elmer, Foster City, Calif.) to amplify longpieces of DNA. This method was developed to allow a single researcher toprocess multiple genes (up to 20 or more) at a time and to obtain anextended (possibly full-length) sequence within 6-10 days. It replacescurrent methods which use labelled probes to screen libraries and allowone researcher to process only about 3-5 genes in 14-40 days.

In the first step, which can be performed in about two days, primers aredesigned and synthesized based on a known partial sequence. In step 2,which takes about six to eight hours, the sequence is extended by PCRamplification of a selected library. Steps 3 and 4, which take about oneday, are purification of the amplified cDNA and its ligation into anappropriate vector. Step 5, which takes about one day, involvestransforming and growing up host bacteria. In step 6, which takesapproximately five hours, PCR is used to screen bacterial clones forextended sequence. The final steps, which take about one day, involvethe preparation and sequencing of selected clones. If the full lengthcDNA has not been obtained, the entire procedure is repeated usingeither the original library or some other preferred library. Thepreferred library may be one that has been size-selected to include onlylarger cDNAs or may consist of single or combined commercially availablelibraries, eg. lung, liver, heart and brain from Gibco/BRL (GaithersburgMd.). The cDNA library may have been prepared with oligo dT or randomprimers. The advantage of using random primed libraries is that theywill have more sequences which contain 5' ends of genes. A randomlyprimed library may be particularly useful if an oligo dT library doesnot yield a complete gene. Obviously, the larger the protein, the lesslikely it is that the complete gene will be found in a single plasmid.

The nucleotide sequence can be used in an assay to detect conditionsassociated with altered expression of CAPE. The nucleotide sequence canbe labeled by methods known in the art and added to a fluid or tissuesample from a patient under hybridizing conditions. After an incubationperiod, the sample is washed with a compatible fluid which optionallycontains a dye (or other label requiring a developer) if the nucleotidehas been labeled with an enzyme. After the compatible fluid is rinsedoff, the dye is quantitated and compared with a standard. If the amountof dye is significantly elevated, the nucleotide sequence has hybridizedwith the sample, and the assay indicates the presence of inflammation,tumor and/or disease.

The nucleotide sequence for cape can be used to construct hybridizationprobes for mapping that gene. The nucleotide sequence provided hereinmay be mapped to a particular chromosome or to specific regions of thatchromosome using well known genetic and/or chromosomal mappingtechniques. These techniques include in situ hybridization, linkageanalysis against known chromosomal markers, hybridization screening withlibraries, flow-sorted chromosomal preparations, or artificialchromosome constructions YAC or P1 constructions. The technique offluorescent in situ hybridization of chromosome spreads has beendescribed, among other places, in Verma et al (1988) Human Chromosomes:A Manual of Basic Techniques, Pergamon Press, New York City.

Fluorescent in situ hybridization of chromosomal preparations and otherphysical chromosome mapping techniques may be correlated with additionalgenetic map data. Examples of genetic map data can be found in the 1994Genome Issue of Science (265:1981f). Correlation between the location ofcape on a physical chromosomal map and a specific disease (orpredisposition to a specific disease) can help delimit the region of DNAassociated with that genetic disease. The nucleotide sequence of thesubject invention may be used to detect differences in gene sequencebetween normal and carrier or affected individuals.

Nucleotide sequences encoding CAPE may be used to produce purified CAPEusing well known methods of recombinant DNA technology. Among the manypublications that teach methods for the expression of genes after theyhave been isolated is Goeddel (1990) Gene Expression Technology, Methodsand Enzymology, Vol 185, Academic Press, San Diego Calif. CAPE may beexpressed in a variety of host cells, either prokaryotic or eukaryotic.Host cells may be from the same species in which cape nucleotidesequences are endogenous or from a different species. Advantages ofproducing CAPE by recombinant DNA technology include obtaining adequateamounts of the protein for purification and the availability ofsimplified purification procedures.

Cells transformed with DNA encoding CAPE may be cultured underconditions suitable for the expression of serpins and recovery of theprotein from the cell culture. CAPE produced by a recombinant cell maybe secreted or may be contained intracellularly, depending on-the capesequence and the genetic construction used. In general, it is moreconvenient to prepare recombinant proteins in secreted form.Purification steps vary with the production process and the particularprotein produced.

In addition to recombinant production, fragments of CAPE may be producedby direct peptide synthesis using solid-phase techniques (cf Stewart etal (1969) Solid-Phase Peptide Synthesis, W H Freeman Co, San FranciscoCalif.; Merrifield J (1963) J Am Chem Soc 85:2149-2154. In vitro proteinsynthesis may be performed using manual techniques or by automation.Automated synthesis may be achieved, for example, using AppliedBiosystems 431A Peptide Synthesizer (Foster City, Calif.) in accordancewith the instructions provided by the manufacturer. Various fragments ofCAPE may be chemically synthesized separately and combined usingchemical methods to produce the full length molecule.

CAPE for antibody induction does not require biological activity;however, the protein must be immunogenic. Peptides used to inducespecific antibodies may have an amino acid sequence consisting of atleast five amino acid, preferably at least 10 amino acid. They shouldmimic an exposed portion of the amino acid sequence of the protein andmay contain the entire amino acid sequence of a small naturallyoccurring molecule such as CAPE. Short stretches of CAPE aa may be fusedwith those of another protein such as keyhole limpet hemocyanin and theresulting chimeric molecule used for antibody production.

Antibodies specific for CAPE may be produced by inoculation of anappropriate animal with the polypeptide or an antigenic fragment. Anantibody is specific for CAPE if it is produced against an epitope ofthe polypeptide and binds to at least part of the natural or recombinantprotein. Antibody production includes not only the stimulation of animmune response by injection into animals, but also analogous steps inthe production of synthetic antibodies or other specific-bindingmolecules such as the screening of recombinant immunoglobulin libraries(Orlandi R et al (1989) PNAS 86:3833-3837, or Huse W D et al (1989)Science 256:1275-1281) or the in vitro stimulation of lymphocytepopulations. Current technology (Winter G and Milstein C (1991) Nature349:293-299) provides for a number of highly specific binding reagentsbased on the principles of antibody formation. These techniques may beadapted to produce molecules specifically binding CAPE.

An additional embodiment of the subject invention is the use of CAPE asa specific protease inhibitor to treat inflammatory or pathologicproblems of the hypothalamus, or of a target tissue. A furtherembodiment of the subject invention is the use of CAPE to specificallybind a small molecule and to modulate its release either within thehypothalamus, a target tissue or extracellularly.

CAPE as a bioactive agent or composition may be administered in asuitable therapeutic dose determined by any of several methodologiesincluding clinical studies on mammalian species to determine maximaltolerable dose and on normal human subjects to determine safe dose.Additionally, the bioactive agent may be complexed with a variety ofwell established compounds or compositions which enhance stability orpharmacological properties such as half-life. It is contemplated thatthe therapeutic, bioactive composition may be delivered by intravenousinfusion into the bloodstream or any other effective means which couldbe used for treating problems involving excess expression and activityof proteases. Alternatively, the compositions may be employed fortreating problems associated with excessive levels of specific smallmolecules.

The examples below are provided to illustrate the subject invention.These examples are provided by way of illustration and are not includedfor the purpose of limiting the invention.

EXAMPLES

I Isolation of mRNA and Construction of cDNA Libraries

The hypothalamic library was constructed from a pooled sample ofhypothalamic tissue taken from the normal human brains of 51 Caucasianmales and females of different ages. The polyadenylated mRNA wasobtained from Clontech Laboratories, Inc. (Catalogue No. #6579-2, PaloAlto Calif.)

The polyadenylated mRNA was used to construct a custom cDNA library(Stratagene, La Jolla Calif.). cDNA synthesis was primed using botholigo dT and random hexamers, and the two cDNA libraries produced weretreated separately. Synthetic adapter oligonucleotides were ligated ontothe cDNA enabling its insertion into the Stratagene Uni-ZAP™ vectorsystem. This system allows high efficiency unidirectional (senseorientation) lambda library construction and the convenience of aplasmid system with blue/white color selection to detect clones withcDNA insertions. Finally, the two cDNA libraries were combined into asingle library by mixing equal numbers of bacteriophage.

The hypothalamic cDNA library can be screened with either DNA probes orantibody probes and the pBluescript® phagemid (Stratagene) can berapidly excised in vivo. The phagemid allows the use of a plasmid systemfor easy insert characterization, sequencing, site directed mutagenesis,creation of unidirectional deletions, and expression of fusion proteins.The custom-constructed library phage particles were infected into E.Coli host strain XL1 Blue® (Stratagene) which has a high transformationefficiency. This efficiency increases the probability of obtaining rare,under-represented clones in the cDNA library. Alternative unidirectionalvectors include but are not limited to pcDNAI (Invitrogen, San DiegoCalif.) and pSHlox-1 (Novagen, Madison Wis.).

II Isolation of cDNA Clones

The phagemid forms of individual cDNA clones were obtained by the invivo excision process, in which XL1-BLUE was coinfected with an f1helper phage. Proteins derived from both lambda phage and f1 helperphage initiated new DNA synthesis from defined sequences on the lambdatarget DNA and create a smaller, single-stranded circular phagemid DNAmolecule that includes all DNA sequences of the pBluescript plasmid andthe cDNA insert. The phagemid DNA was released from the cells andpurified, then used to reinfect fresh bacterial host cells (SOLR,Stratagene Inc), where the double-stranded phagemid DNA was produced.Because the phagemid carries the gene for β-lactamase, the newlytransformed bacteria were selected on medium containing ampicillin.

Phagemid DNA was purified using the QIAWELL-8 Plasmid PurificationSystem® (QIAGEN Inc, Chatsworth Calif.). This technique provides a rapidand reliable high-throughput method for lysing the bacterial cells andisolating highly purified phagemid DNA. The DNA eluted from thepurification resin was suitable for DNA sequencing and other analyticalmanipulations.

An alternate method of purifying phagemid has recently become available.It utilizes the Miniprep Kit (Catalog No. #77468, Advanced GeneticTechnologies Corporation, Gaithersburg Md.). This kit is in the 96-wellformat and provides enough reagents for 960 purifications. Each kit isprovided with a recommended protocol, which has been employed except forthe following changes. First, the 96 wells are each filled with only 1ml of sterile terrific broth with carbenicillin at 25 mg/L and glycerolat 0.4%. After the wells are inoculated, the bacteria are cultured for24 hours and lysed with 60 μl of lysis buffer. A centrifugation step(2900 rpm for 5 minutes) is performed before the contents of the blockare added to the primary filter plate. The optional step of addingisopropanol to TRIS buffer is not routinely performed. After the laststep in the protocol, samples are transferred to a Beckman 96-well blockfor storage.

III Sequencing of cDNA Clones

The cDNA inserts from random isolates of the hypothalamus library weresequenced in part. Methods for DNA sequencing are well known in the art.Conventional enzymatic methods employed DNA polymerase Klenow fragment,SEQUENASE® (US Biochemical Corp, Cleveland, Ohio) or Taq polymerase toextend DNA chains from an oligonucleotide primer annealed to the DNAtemplate of interest. Methods have been developed for the use of bothsingle- and double-stranded templates. The chain termination reactionproducts were electrophoresed on urea-acrylamide gels and detectedeither by autoradiography (for radionuclide-labeled precursors) or byfluorescence (for fluorescent-labeled precursors). Recent improvementsin mechanized reaction preparation, sequencing and analysis using thefluorescent detection method have permitted expansion in the number ofsequences that can be determined per day (using machines such as theCatalyst 800 and the Applied Biosystems 377 or 373 DNA sequencer).

IV Homology Searching of cDNA Clones and Deduced Proteins

Each sequence so obtained was compared to sequences in GenBank using asearch algorithm developed by Applied Biosystems Inc. and incorporatedinto its INHERIT™ 670 Sequence Analysis System. In this algorithm,Pattern Specification Language (developed by TRW Inc.) was used todetermine regions of homology. The three parameters that determine howthe sequence comparisons run were window size, window offset, and errortolerance. Using a combination of these three parameters, the DNAdatabase was searched for sequences containing regions of homology tothe query sequence, and the appropriate sequences were scored with aninitial value. Subsequently, these homologous regions were examinedusing dot matrix homology plots to distinguish regions of homology fromchance matches. Smith-Waterman alignments of the protein sequence wereused to display the results of the homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT 670 Sequence Analysis System in a way similar to that used inDNA sequence homologies. Pattern Specification Language and parameterwindows were used to search protein databases for sequences containingregions of homology which were scored with an initial value. Dot-matrixhomology plots were examined to distinguish regions of significanthomology from chance matches.

Alternatively, BLAST, which stands for Basic Local Alignment SearchTool, is used to search for local sequence alignments (Altschul, S F(1993) J. Mol Evol 36:290-300; Altschul, S F et al (1990) J Mol Biol215:403-10). BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologues. Although it is ideal for matcheswhich do not contain gaps, it is inappropriate for performingmotif-style searching. The fundamental unit of BLAST algorithm output isthe high-scoring segment pair (HSP).

An HSP consists of two sequence fragments of arbitrary but equal lengthswhose alignment is locally maximal and for which the alignment scoremeets or exceeds a threshold or cutoff score set by the user. The BLASTapproach is to look for HSPs between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. The parameter E establishes the statisticallysignificant threshold for reporting database sequence matches. E isinterpreted as the upper bound of the expected frequency of chanceoccurrence of an HSP (or set of HSPs) within the context of the entiredatabase search. Any database sequence whose match satisfies E isreported in the program output.

V Identification and Full Length Sequencing of the Genes

The nucleotide sequence for the entire coding region of thehypothalamus-derived serpin, CAPE, claimed in this invention is shown inFIG. 1. The CDNA of Incyte 84476 was extended to full length using amodified XL-PCR (Perkin Elmer) procedure. Primers were designed based onknown sequence; one primer was synthesized to initiate extension in theantisense direction (XLR) and the other to extend sequence in the sensedirection (XLF). The sequences of these primers and their location areas follows: XLR (nucleotides 502-525 in SEQ ID NO:1) and XLF(nucleotides 609-632 in SEQ ID NO:1) . The primers allowed the sequenceto be extended "outward" generating amplicons containing new, unknownnucleotide sequence for the gene of interest. The primers were designedusing Oligo 4.0 (National Biosciences Inc, Plymouth Minn.) to be 22-30nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures about 68°-72° C. Anystretch of nucleotides which would result in hairpin structures andprimer primer dimerizations were avoided.

The hypothalamus cell cDNA library was used as a template, and XLR andXLF primers were used to extend and amplify the 84476 sequence. Byfollowing the instructions for the XL-PCR kit, the enzymes provided highfidelity in the amplification. Beginning with 25 pMol of each primer andthe recommended concentrations of all other components of the kit, PCRwas performed using the MJ PTC200 (MJ Research, Watertown Mass.) and thefollowing parameters:

    ______________________________________    Step 1       94° C. for 60 sec (initial denaturation)    Step 2       94° C. for 15 sec    Step 3       65° C. for 1 min    Step 4       68° C. for 7 min    Step 5       Repeat step 2-4 for 15 additional cycles    Step 6       94° C. for 15 sec    Step 7       65° C. for 1 min    Step 8       68° C. for 7 min + 15 sec/cycle    Step 9       Repeat step 6-8 for 11 additional cycles    Step 10      72° C. for 8 min    Step 11      4° C. (and holding)    ______________________________________

At the end of 28 cycles, 50 μl of the reaction mix was removed; and theremaining reaction mix was run for an additional 10 cycles as outlinedbelow:

    ______________________________________    Step 1       94° C. for 15 sec    Step 2       65° C. for 1 min    Step 3       68° C. for (10 min + 15 sec)/cycle    Step 4       Repeat step 1-3 for 9 additional cycles    Step 5       72° C. for 10 min    ______________________________________

A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration, about 0.6-0.8%, agarose mini-gelto determine which reactions were successful in extending the sequence.Although all extensions potentially contain a full length gene, some ofthe largest products or bands were selected and cut out of the gel.Further purification involved using a commercial gel extraction methodsuch as QIAQuick™ (QIAGEN Inc, Chatsworth Calif.). After recovery of theDNA, Klenow enzyme was used to trim single stranded, nucleotideoverhangs creating blunt ends which facilitated religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer. Then, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook J et al, supra).After incubation for one hour at 37° C., the whole transformationmixture was plated on Luria Bertani (LB)-agar (Sambrook J et al, supra)containing carbenicillin at 25 mg/L. The following day, 12 colonies wererandomly picked from each plate and cultured in 150 μl of liquidLB/carbenicillin medium placed in an individual well of an appropriate,commercially-available, sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture was transferred into a non-sterile96-well plate and after dilution 1:10 with water, 5 μl of each samplewas transferred into a PCR array.

For PCR amplification, 15 μl of PCR mix (1.33× containing 0.75 units ofTaq polymerase, a vector primer and one or both of the gene specificprimers used for the extension reaction) were added to each well.Amplification was performed using the following conditions:

    ______________________________________    Step 1     94° C. for 60 sec    Step 2     94° C. for 20 sec    Step 3     55° C. for 30 sec    Step 4     72° C. for 90 sec    Step 5     Repeat steps 2-4 for an additional 29 cycles    Step 6     72° C. for 180 sec    Step 7     4° C. (and holding)    ______________________________________

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid and sequenced.

VI Antisense analysis

Knowledge of the cDNA sequence of the novel serpin gene will enable itsuse in antisense technology in the investigation of gene function.Oligonucleotides, genomic or cDNA fragments comprising the antisensestrand of cape can be used either in vitro or in vivo to inhibitexpression of the protein. Such technology is now well known in the art,and probes can be designed at various locations along the nucleotidesequence. By transfection of cells or whole test animals with suchantisense sequences, the gene of interest can effectively be turned off.Frequently, the function of the gene can be ascertained by observingbehavior at the cellular, tissue or organismal level (e.g. lethality,loss of differentiated function, changes in morphology, etc).

In addition to using sequences constructed to interrupt transcription ofthe open reading frame, modifications of gene expression can be obtainedby designing antisense sequences to intron regions, promoter/enhancerelements, or even to trans-acting regulatory genes. Similarly,inhibition can be achieved using Hogeboom base-pairing methodology, alsoknown as "triple helix" base pairing.

VII Expression of CAPE

Expression of CAPE may be accomplished by subcloning the cDNAs intoappropriate expression vectors and transfecting the vectors intoappropriate expression hosts. In this particular case, the cloningvector used in the generation of the full length clone also provides fordirect expression of the included cape sequence in E. coli. Upstream ofthe cloning site, this vector contains a promoter for β-galactosidase,followed by sequence containing the amino-terminal Met and thesubsequent 7 residues of β-galactosidase. Immediately following theseeight residues is an engineered bacteriophage promoter useful forartificial priming and transcription and for providing a number ofunique endonuclease restriction sites for cloning.

Induction of the isolated, transfected bacterial strain with IPTG usingstandard methods will produce a fusion protein corresponding to thefirst seven residues of β-galactosidase, about 15 residues of "linker",and the peptide encoded within the cDNA. Since cDNA clone inserts aregenerated by an essentially random process, there is one chance in threethat the included cDNA will lie in the correct frame for propertranslation. If the cDNA is not in the proper reading frame, it can beobtained by deletion or insertion of the appropriate number of bases bywell known methods including in vitro mutagenesis, digestion withexonuclease III or mung bean nuclease, or oligonucleotide linkerinclusion.

The cape cDNA can be shuttled into other vectors known to be useful forexpression of protein in specific hosts. Oligonucleotide amplimerscontaining cloning sites as well as a segment of DNA sufficient tohybridize to stretches at both ends of the target cDNA (25 bases) can besynthesized chemically by standard methods. These primers can then beused to amplify the desired gene segments by PCR. The resulting new genesegments can be digested with appropriate restriction enzymes understandard conditions and isolated by gel electrophoresis. Alternately,similar gene segments can be produced by digestion of the cDNA withappropriate restriction enzymes and filling in the missing gene segmentswith chemically synthesized oligonucleotides. Segments of the codingsequence from more than one gene can be ligated together and cloned inappropriate vectors to optimize expression of recombinant sequence.

Suitable expression hosts for such chimeric molecules include but arenot limited to mammalian cells such as Chinese Hamster Ovary (CHO) andhuman 293 cells, insect cells such as Sf9 cells, yeast cells such asSaccharomyces cerevisiae, and bacteria such as E. coli. For each ofthese cell systems, a useful expression vector may also include anorigin of replication to allow propagation in bacteria and a selectablemarker such as the β-lactamase antibiotic resistance gene to allowselection in bacteria. In addition, the vectors may include a secondselectable marker such as the neomycin phosphotransferase gene to allowselection in transfected eukaryotic host cells. Vectors for use ineukaryotic expression hosts may require RNA processing elements such as3' polyadenylation sequences if such are not part of the cDNA ofinterest.

Additionally, the vector may contain promoters or enhancers whichincrease gene expression. Such promoters are host specific and includeMMTV, SV40, or metallothionine promoters for CHO cells; trp, lac, tac orT7 promoters for bacterial hosts, or alpha factor, alcohol oxidase orPGH promoters for yeast. Transcription enhancers, such as the roussarcoma virus (RSV) enhancer, may be used in mammalian host cells. Oncehomogeneous cultures of recombinant cells are obtained through standardculture methods, large quantities of recombinantly produced CAPE can berecovered from the conditioned medium and analyzed using chromatographicmethods known in the art.

VIII Isolation of Recombinant CAPE

CAPE may be expressed as a chimeric protein with one or more additionalpolypeptide domains added to facilitate protein purification. Suchpurification facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp., SeattleWash.). The inclusion of a cleavable linker sequence such as Factor XAor enterokinase (Invitrogen) between the purification domain and thecape sequence may be useful to facilitate expression of CAPE.

IX Production of CAPE Specific Antibodies

Two approaches are utilized to raise antibodies to CAPE, and eachapproach is useful for generating either polyclonal or monoclonalantibodies. In one approach, denatured protein from the reverse phaseHPLC separation is obtained in quantities up to 75 mg. This denaturedprotein can be used to immunize mice or rabbits using standardprotocols; about 100 micrograms are adequate for immunization of amouse, while up to 1 mg might be used to immunize a rabbit. Foridentifying mouse hybridomas, the denatured protein can beradioiodinated and used to screen potential murine B-cell hybridomas forthose which produce antibody. This procedure requires only smallquantities of protein, such that 20 mg would be sufficient for labelingand screening of several thousand clones.

In the second approach, the amino acid sequence of CAPE, as deduced fromtranslation of the cDNA, is analyzed to determine regions of highimmunogenicity. Oligopeptides comprising regions which are hydrophilic,highly antigenic, or highly likely to be on the serpin surface, as shownin FIG. 3, are synthesized and used in suitable immunization protocolsto raise antibodies. Analysis to select appropriate epitopes isdescribed by Ausubel F M et al (supra). The optimal amino acid sequencesfor immunization are usually at the C-terminus, the N-terminus and thoseintervening, hydrophilic regions of the polypeptide which are likely tobe exposed to the external environment when the protein is in itsnatural conformation.

Typically, selected peptides, about 15 residues in length, aresynthesized using an Applied Biosystems Peptide Synthesizer Model 431Ausing fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH) byreaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; cf.Ausubel F M et al, supra). If necessary, a cysteine may be introduced atthe N-terminus of the peptide to permit coupling to KLH. Rabbits areimmunized with the peptide-KLH complex in complete Freund's adjuvant.The resulting antisera are tested for antipeptide activity by bindingthe peptide to plastic, blocking with 1% BSA, reacting with antisera,washing and reacting with labeled (radioactive or fluorescent), affinitypurified, specific goat anti-rabbit IgG.

Hybridomas may also be prepared and screened using standard techniques.Hybridomas of interest are detected by screening with labeled CAPE toidentify those fusions producing the monoclonal antibody with thedesired specificity. In a typical protocol, wells of plates (FAST;Becton-Dickinson, Palo Alto Calif.) are coated with affinity purified,specific rabbit-anti-mouse (or suitable anti-species lg) antibodies at10 mg/ml. The coated wells are blocked with 1% BSA, washed and exposedto supernatants from hybridomas. After incubation the wells are exposedto labeled CAPE, 1 mg/ml. Clones producing antibodies will bind aquantity of labeled CAPE which is detectable above background. Suchclones are expanded and subjected to 2 cycles of cloning at limitingdilution (1 cell/3 wells). Cloned hybridomas are injected into pristinemice to produce ascites, and monoclonal antibody is purified from mouseascitic fluid by affinity chromatography on Protein A. Monoclonalantibodies with affinities of at least 10⁸ M⁻¹, preferably 10⁹ to 10¹⁰or stronger, will typically be made by standard procedures as describedin Harlow and Lane (1988) Antibodies: A Laboratory Manual. Cold SpringHarbor Laboratory, Cold Spring Harbor N.Y.; and in Goding (1986)Monoclonal Antibodies: Principles and Practice, Academic Press, New YorkCity, both incorporated herein by reference.

X Diagnostic Test Using CAPE Specific Antibodies

Particular CAPE antibodies are useful for the diagnosis of prepathologicconditions, and chronic or acute diseases which are characterized bydifferences in the amount or distribution of CAPE. To date, CAPE hasonly been found to be expressed in a hypothalamus library and thus maybe specific for conditions that damage the hypothalamus and which canthen be detected.

Diagnostic tests for CAPE include methods utilizing the antibody and alabel to detect CAPE in human body fluids, tissues or extracts of suchtissues. The polypeptides and antibodies of the present invention may beused with or without modification. Frequently, the polypeptides andantibodies will be labeled by joining them, either covalently ornoncovalently, with a substance which provides for a detectable signal.A wide variety of labels and conjugation techniques are known and havebeen reported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent agents, chemiluminescent agents, chromogenicagents, magnetic particles and the like. Patents teaching the use ofsuch labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinantimmunoglobulins may be produced as shown in U.S. Pat. No. 4,816,567,incorporated herein by reference.

A variety of protocols for measuring soluble or membrane-bound CAPE,using either polyclonal or monoclonal antibodies specific for therespective protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). A two-site monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson CAPE is preferred, but a competitive binding assay may be employed.These assays are described, among other places, in Maddox, Del. et al(1983, J Exp Med 158:1211).

XI Purification of Native CAPE Using Specific Antibodies

Native or recombinant CAPE can be purified by immunoaffinitychromatography using antibodies specific for CAPE. In general, animmunoaffinity column is constructed by covalently coupling theanti-CAPE antibody to an activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated Sepharose (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such immunoaffinity columns are utilized in the purification of CAPE bypreparing a fraction from cells containing CAPE in a soluble form. Thispreparation is derived by solubilization of the whole cell or of asubcellular fraction obtained via differential centrifugation by theaddition of detergent or by other methods well known in the art.Alternatively, soluble CAPE containing a signal sequence may be secretedin useful quantity into the medium in which the cells are grown.

A soluble CAPE-containing preparation is passed over the immunoaffinitycolumn, and the column is washed under conditions that allow thepreferential absorbance of serpin (eg, high ionic strength buffers inthe presence of detergent). Then, the column is eluted under conditionsthat disrupt antibody/CAPE binding (e.g., a buffer of pH 2-3 or a highconcentration of a chaotrope such as urea or thiocyanate ion), and CAPEis collected.

XII CAPE Activity

The activity of purified or expressed CAPE in protease inhibition may betested by mixing a known quantity of the enzyme with a potentialsubstrate protease such as chymotrypsin and a purified protein whichchymotrypsin usually cleaves. The ability of a given amount of CAPE toinhibit chymotrypsin can be assayed by FPLC of the protein fragmentsproduced under a given set of conditions in a specific period of time.

In another method to test CAPE activity as a protease inhibitor, asample of the reaction materials may be run on a nondenaturing gel whichseparates the protease inhibitor complex, protease, inhibitor, proteinsubstrate and protein fragments as different sized peptides.

The activity of purified or expressed CAPE in small molecule binding maybe tested by incubating CAPE with various small molecules, preferablythose derived from the hypothalamus or those that affect hypothalamusfunction, in radiolabeled form. After allowing a suitable time forbinding, CAPE-bound small molecules may be separated from free smallmolecules by FPLC, and the binding affinity of CAPE for different smallmolecules determined.

XIII Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of small molecules withwhich they interact, eg, inhibitors, agonists, antagonists, etc. Any ofthese examples can be used to fashion drugs which are more active orstable forms of the polypeptide or which enhance or interfere with thefunction of a polypeptide in vivo (Hodgson J (1991) Bio/Technology9:19-21, incorporated herein by reference).

In one approach, the three-dimensional structure of a protein ofinterest, or of a protein-inhibitor complex, is determined by x-raycrystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of thepolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of a polypeptide may be gained by modeling basedon the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous serpin-likemolecules, to identify efficient inhibitors, or to identify smallmolecules that may bind serpins. Useful examples of rational drug designmay include molecules which have improved activity or stability as shownby Braxton S and Wells J A (1992 Biochemistry 31:7796-7801) or which actas inhibitors, agonists, or antagonists of native peptides as shown byAthauda S B et al (1993 J Biochem 113:742-746), incorporated herein byreference.

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amount of polypeptide maybe made available to perform such analytical studies as X-raycrystallography. In addition, knowledge of the CAPE amino acid sequenceprovided herein will provide guidance to those employing computermodeling techniques in place of or in addition to x-ray crystallography.

XIV Use and Administration of CAPE

Since CAPE may be a protease inhibitor, it may be used to treat tissuewasting associated with excessive protease production duringinflammation or diseases associated with nervous tissue degeneration.The tissues that may be affected by wasting may be the hypothalamus,where the serpin is expressed or tissues surrounding or adjacent to thehypothalamus. For example, neuronal loss in the NTL associated withdiseases such as Kallmann's and Down's syndromes, or in Alzheimer's andHuntington's diseases may be prevented by administration of CAPE.Destruction of the posterior hypothalamus by ischemia, encephalitis,trauma or tumor may also be prevented.

On the other hand, CAPE may be a small molecule binding protein whichcan be used to modulate levels of specific small molecules in thetreatment of disease. For example, anorexia, bulimia, depression, andsome forms of diabetes may be related to the overproduction of one ormore of the molecules, such as CRH, ACTH, TRH, TSH, GRH, GH, insulin,somatostatin, cholecystokinin, interleukins, oxytocin, insulin-likegrowth factors, glucagon, etc., which govern the nonendocrine intake andeating behaviors. CAPE may be employed to bind one of these molecules,thereby decreasing the symptoms of these diseases.

CAPE may also be used to decrease the amount of free circulatingsomatostatin to prevent somatostatin's inhibitory effect on the releaseof growth hormone. In another example, if CAPE were to remove excesslevels of circulating prolactin, diseases such as galactorrhea and/orhypogandism may be avoided. Therefore, administration of CAPE may beuseful to sequester some of these small molecules to prevent disease.

CAPE will be formulated in a nontoxic, inert, pharmaceuticallyacceptable aqueous carrier medium (CAPE treatment, CT) preferably at apH of about 5 to 8, more preferably 6 to 8, although the pH may varyaccording to the characteristics of the formulation and itsadministration. Characteristics such as solubility of the molecule,half-life and antigenicity/immunogenicity will aid in defining aneffective carrier. Native human proteins are preferred as CT, butrecombinant, organic or synthetic molecules resulting from drug designmay be equally effective in particular situations.

CTs may be delivered by known routes of administration including but notlimited to transmucosal spray and aerosol, transdermal patch andbandage, intravenous formulations, orally administered liquids and pillsparticularly formulated to resist stomach acid and enzymes. For higherspecificity in administration, CTs may be directly injected or implantedin the brain, close to the hypothalamus.

The particular formulation, exact dosage, and route of administrationwill be determined by the attending physician and will vary according toeach specific situation. Such determinations are made by consideringmultiple variables such as the condition to be treated, the CT to beadministered, and the pharmacokinetic profile of the particular CT.Additional factors which may be taken into account include disease state(e.g. severity) of the patient, age, weight, gender, diet, time ofadministration, drug combination, reaction sensitivities, andtolerance/response to therapy. Long acting CT formulations might beadministered every 3 to 4 days, every week, or once every two weeksdepending on half-life and clearance rate of the particular CT.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature; see U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.It is anticipated that different formulations will be effective fordifferent uses of CT and that administration targeting a tissue or organmay necessitate delivery in a specific manner.

All publications and patents mentioned in the above specification areherein incorporated by reference. The foregoing written specification isconsidered to be sufficient to enable one skilled in the art to practicethe invention. Indeed, various modifications of the above describedmodes for carrying out the invention which are readily apparent to thoseskilled in the field of molecular biology or related fields are intendedto be within the scope of the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 5    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1558 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: Hypothalamus              (B) CLONE: 84476    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    #GCCAATCAGG    60GGAAAGG AGAGGAAGGG GGGGGCAAGC CCTCACCTGC    #TCTGCAGAGT   120ACAATAT GGCTTTCCTG GGACTCTTCT CTTTGCTGGT    #GAATATGTAT   180CCACTTT CCCTGAGGAA GCCATTGTTG ACTTGTCAGT    #GAGTATTGCT   240CCACTGG TGAAGATGAA AATATTCTCT TCTCTCCATT    #AATCCGCCAC   300TGATGGA ACTTGGGGCC CAAGGATCTA CCCAGAAAGA    #GGAGTTTTCA   360ACAGCCT AAAAAATGGT GAAGAATTTT CTTTCTTGAA    #CTTGTTTGTG   420CTAAAGA GAGCCAATAT GTGATGAAAA TTGCCAATTC    #TTTTAATGCA   480ATGTCAA TGAGGAGTTT TTGCAAATGA TGAAAAAATA    #CAATAAGTGG   540TGGACTT CAGTCAAAAT GTAGCCGTGG CCAACTACAT    #TTTTNATGCT   600CAAACAA TCTGGTGAAA GATTTGGTAT CCCCAAGGGA    #GTCGCAGTTT   660CCCTCAT TAATGCTGTC TATTTCAAGG GGAACTGGAA    #AGTCCAAATT   720CTAGAAC CTTTTCTTTC ACTAAAGATG ATGAAAGTGA    #CTCCAATGAA   780AGCAAGG AGAATTTTAT TATGGGGAAT TTAGTGATGG    #AAGCATGATG   840ACCAAGT CCTAGAAATA CCATATGAAG GAGATGAAAT    #CAAAGCACAG   900GACAGGA AGTTCCTCTT GCTACTCTGG AGCCATTAGT    #CCTGCCCAGG   960GGGCAAA CTCTGTGAAG AAGCAAAAAG TAGAAGTATA    #AATAACTGAA  1020AGGAAAT TGATTTAAAA GATGTTTTGA AGGCTCTTGG    #TCTTTCCAAA  1080TCAAATT TGACAGCCTC TCTGATAATA AGGAGATTTT    #TGTCTCAGGA  1140CCTTCCT AGAGGTTAAT GAAGAAGGCT CAGAACTCTC    #TCCATTTTTC  1200TAGGATG CTGTCTGTAT CCTCAAGTTA TTGTCGACCA    #CATGCATCCT  1260ACAGGAG AACTGGTACA ATTCTATTCA TGGGACGAGT    #ATTTGAATAA  1320CAAGTGG ACATGATTTC GAAGAACTTT AAGTTACTTT    #GGATTTGTGT  1380AACTAAG CACATTATGT TTGCAACTGG TATATATTTA    #TATATGTAAA  1440TTAAGAT AATATTTAAA ATAGTTCCAG ATAAAAACAA    #TTATGTCATT  1500GTCAAGG AATGTTATCA GTATTAAGCT AATGGTCCTG    #AAAAAAAA    1558GTTGTTT AAAATAAAAG TACCTATTGA AAAAAAAAAA    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 407 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    #Val Leu Gln Ser Met Alaeu Phe Ser Leu Leu    #                 15    #Val Asp Leu Ser Val Asnro Glu Glu Ala Ile    #             30    #Asp Glu Asn Ile Leu Pherg Ala Thr Gly Glu    #         45    #Met Met Glu Leu Gly Alala Leu Ala Met Gly    #     60    #Ser Met Gly Tyr Asp Serys Glu Ile Arg His    # 80    #Lys Glu Phe Ser Asn Metlu Phe Ser Phe Leu    #                 95    #Lys Ile Ala Asn Ser Leuer Gln Tyr Val Met    #            110    #Glu Phe Leu Gln Met Methe His Val Asn Glu    #        125    #Val Asp Phe Ser Gln Asnla Ala Val Asn His    #    140    #Val Glu Asn Asn Thr Asnyr Ile Asn Lys Trp    #160    #Asp Phe Xaa Ala Ala Threu Val Ser Pro Arg    #                175    #Lys Gly Asn Trp Lys Sersn Ala Val Tyr Phe    #            190    #Ser Phe Thr Lys Asp Aspsn Thr Arg Thr Phe    #        205    #Gln Gln Gly Glu Phe Tyrle Pro Met Met Tyr    #    220    #Ala Gly Gly Ile Tyr Glnsp Gly Ser Asn Glu    #240    #Ile Ser Met Met Leu Valyr Glu Gly Asp Glu    #                255    #Leu Glu Pro Leu Val Lysal Pro Leu Ala Thr    #            270    #Val Lys Lys Gln Lys Vallu Trp Ala Asn Ser    #        285    #Gln Glu Ile Asp Leu Lysrg Phe Thr Val Glu    #    300    #Ile Phe Ile Lys Ile Lyseu Gly Ile Thr Glu    #320    #Phe Leu Ser Lys Ala Ilesp Asn Lys Glu Ile    #                335    #Gly Ser Glu Leu Ser Vallu Val Asn Glu Glu    #            350    #Leu Tyr Pro Gln Val Ileeu Val Gly Cys Cys    #        365    #Asn Arg Arg Thr Gly Thrhe Phe Leu Ile Arg    #    380    #Glu Thr Met Asn Thr Serrg Val Met His Pro    #400    -  Gly His Asp Phe Glu Glu Leu                     405    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 382 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -      (v) FRAGMENT TYPE: N-terminal    -     (vi) ORIGINAL SOURCE:    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #Phe Ala Leu Asn Leu Pheal Ala Asn Thr Leu    #                 15    #Asn Leu Phe Leu Ser Prola Ser Pro Thr Gln    #             30    #Tyr Met Gly Ser Arg Glyhr Met Ala Met Val    #         45    #Gln Phe Asn Glu Val Glyet Ala Lys Val Leu    #     60    #Arg Ser Leu Ser Ser Alale His Ser Ser Phe    # 80    #Glu Ser Val Asn Lys Leuly Asn Tyr Leu Leu    #                 95    #Glu Tyr Ile Arg Leu Cysla Ser Phe Arg Glu    #            110    #Val Asp Phe Leu Glu Cyser Glu Pro Gln Ala    #        125    #Trp Val Lys Thr Gln Thrys Lys Ile Asn Ser    #    140    #Gly Ser Val Asp Gly Aspsn Leu Leu Pro Glu    #160    #Phe Lys Gly Lys Trp Lysal Asn Ala Val Tyr    #                175    #Tyr Pro Phe Arg Val Asnys Leu Asn Gly Leu    #            190    #Tyr Leu Arg Glu Lys Leuro Val Gln Met Met    #        205    #Gln Ile Leu Glu Leu Prolu Asp Leu Lys Ala    #    220    #Leu Pro Asp Glu Ile Alaer Met Phe Leu Leu    #240    #Ser Glu Ile Thr Tyr Aspeu Glu Leu Leu Glu    #                255    #Met Ala Glu Asp Glu Valhr Ser Lys Asp Lys    #            270    #Glu His Tyr Glu Leu Argln Phe Lys Leu Glu    #        285    #Ala Phe Asn Lys Gly Arget Gly Met Glu Asp    #    300    #Asp Leu Phe Leu Ser Gluet Ser Glu Arg Asn    #320    #Glu Glu Gly Thr Glu Alaet Val Asp Val Asn    #                335    #Arg Thr Gly His Gly Glyly Val Met Thr Gly    #            350    #Phe Leu Ile Met His Lyssp His Pro Phe Leu    #        365    #Phe Ser Ser Proys Ile Leu Phe Phe Gly Arg    #    380    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 420 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -      (v) FRAGMENT TYPE: N-terminal    -     (vi) ORIGINAL SOURCE:    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    #Val Leu Glu Ser Leu Alaeu Phe Ser Leu Leu    #                 15    #Val Asp Leu Ala Val Asnro Glu Glu Ala Ile    #             30    #Thr Glu Asn Leu Leu Leula Ala Ala Gly Glu    #         45    #Met Val Glu Leu Gly Alala Leu Ala Met Gly    #     60    #Val Leu Gly Phe Asp Sersp Glu Ile Ala Lys    # 80    #Ser Leu Lys Ser Leu Sersp Lys Ile His Ser    #                 95    #Val Leu Glu Ile Ala Asner Thr Gly Asn Tyr    #            110    #Asn Glu Glu Phe Leu Glnsn Gly Ala Ser Val    #        125    #Asn Ala Val Asp Phe Leuhe Ser Ala Ala Val    #    140    #Asn Ser Trp Val Glu Thrla Arg Asn Lys Ile    #160    #Ser Glu Gly Ser Val Xaaal Lys Asp Leu Val    #                175    #Val Tyr Phe Lys Gly Asnla Leu Val Asn Ala    #            190    #Gly Leu Phe Ser Phe Thrlu Lys Glu Leu Thr    #        205    #Met Met Tyr Leu Gln Glylu Val Gln Val Gln    #    220    #Leu Asn Ala Ala Gly Glylu Phe Ile Asp Gly    #240    #Gly Asp Glu Val Ser Metlu Leu Pro Tyr Ala    #                255    #Val Ala Thr Gly Leu Glusp Glu Ile Ala Asp    #            270    #Val Glu Glu Trp Ala Seral Thr Ala Asp Leu    #        285    #Val Tyr Leu Pro Gln Phelu Asp Glu Val Glu    #    300    #Val Leu Lys Ala Leu Glyle Asp Leu Lys Ser    #320    #Asn Phe Ser Gly Leu Serle Lys Gly Lys Ala    #                335    #Ile His Gln Ala Phe Valhe Leu Ser Glu Ala    #            350    #Ala Gly Gly Gly Gly Vally Ser Glu Ala Ala    #        365    #Gln Val Val Ala Asp Hisly His Gly Gly Pro    #    380    #Thr Gly Thr Ile Leu Phele Arg Asn Lys Ile    #400    #Asn Thr Ser Gly His Aspis Pro Glu Thr Met    #                415    -  Phe Ser Ser Leu                 420    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 406 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -      (v) FRAGMENT TYPE: N-terminal    -     (vi) ORIGINAL SOURCE:    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    #Ala Ile Val Leu Leu Alaal Phe Pro Glu Glu    #                 15    #Gly Glu Thr Glu Asn Leuis Leu Ala Ala Ala    #             30    #Met Gly Met Val Glu Leuer Ile Ala Leu Ala    #         45    #Ala Lys Val Leu Gly Phehr Glu Asp Glu Ile    #     60    #His Ser Ser Leu Lys Serly Ala Asp Lys Ile    # 80    #Asn Tyr Val Leu Glu Ilehr Ala Ser Thr Gly    #                 95    #Ser Val Asn Glu Glu Phely Glu Asn Gly Ala    #            110    #Ala Val Asn Ala Val Aspys Tyr Phe Ser Ala    #        125    #Lys Ile Asn Ser Trp Valla Val Ala Arg Asn    #    140    #Leu Val Ser Glu Gly Serly Leu Val Lys Asp    #160    #Asn Ala Val Tyr Phe Lysrg Leu Ala Leu Val    #                175    #Leu Thr Gly Leu Phe Serln Phe Glu Lys Glu    #            190    #Val Gln Met Met Tyr Leula Ser Glu Val Gln    #        205    #Asp Gly Leu Asn Ala Alale Gly Glu Phe Ile    #    220    #Tyr Ala Gly Asp Glu Valal Leu Glu Leu Pro    #240    #Ala Asp Val Ala Thr Glyeu Ser Asp Glu Ile    #                255    #Asp Leu Val Glu Glu Trper Leu Val Thr Ala    #            270    #Val Glu Val Tyr Leu Proys Ala Glu Asp Glu    #        285    #Lys Ser Val Leu Lys Alalu Glu Ile Asp Leu    #    300    #Lys Ala Asn Phe Ser Glyla Phe Ile Lys Gly    #320    #Glu Ala Ile His Gln Alasp Leu Phe Leu Ser    #                335    #Ala Ala Ala Gly Gly Glylu Glu Gly Ser Glu    #            350    #Gly Pro Gln Val Val Alarg Thr Gly His Gly    #        365    #Lys Ile Thr Gly Thr Ilehe Leu Ile Arg Asn    #    380    #Thr Met Asn Thr Ser Glyal Met His Pro Glu    #400    -  His Asp Phe Ser Ser Leu                     405    __________________________________________________________________________

We claim:
 1. A purified CAPE polypeptide whose amino acid sequence isshown in SEQ ID NO
 2. 2. A purified CAPE polypeptide having the aminoacid sequence shown as residues 51-519 of SEQ ID NO:2.